DNA in the Cell chromosome cell nucleus Double

DNA in the Cell chromosome cell nucleus Double stranded DNA molecule Target Region for PCR Individual nucleotides Polymerase Chain Reaction. what is it? Invented 1990; Nobel Prize in 1993: Kary Mullis PCR, stands for?

DNA Amplification with the Polymerase Chain Reaction (PCR) 5’ 3’ 3’ 3’ 5’ 5’ Starting DNA Template Separate strands (denature) Forward primer 5’ 3’ Make copies Add primers (extend primers) 5’ (anneal) 3’ 3’ 5’ Reverse primer

PCR (Polymerase Chain Reaction) Copies DNA Exponentially through Multiple Thermal Cycles Original DNA target region Heat Oligo’s 1 copy DNA Polymerase d. NTP Cool In 32 cycles at 100% efficiency, Heat Cool 2 copies 4 copies Heat … ? ? billion copies are created 1. 07 To work, what property of DNA polymerase have to have? New bugsinland up polymerase in court. . . Microorganisms hot Heat. Scientists stable (1998)…Yellowstone's so don’t have to add new for everyfrom cycle springs are especially valuable because their enzymes are not easily destroyed by heat. . Thermostable organisms, e. g. living in Yellowstone Geysers have this.

Forensic DNA Typing or Did you kill (rape, father…) that person? How DNA can “definitively” say. Adapted from: National Institutes of Science & Technology http: //www. cstl. nist. gov/div 831/strbase/intro. htm

DNA Use in Forensic, Paternity… Cases • Most Forensic cases are rape cases (>2 out of 3) Looking for match between evidence and suspect -- matching suspect with evidence • Paternity testing -- identifying father • Military DNA “dog-tag. ” Challenges • Mixtures must be resolved • DNA is often degraded (stored wet- have mold, nuclease) • Inhibitors to PCR are often present

Sources of Biological Evidence • • Blood Semen Saliva Urine Hair Teeth (useful in fires). Bone (Yes, there are cells in bone. Decalcify it. 100, 000 year old people, Dinosaurs- has DNA!) • Tissue All felony arrests- cheek swab.

Short Tandem Repeats (STRs) (say chromo 3) Identical in all people AATG 7 repeats Identical in all people 8 repeats the repeat region is variable between samples while the flanking regions where PCR primers bind are constant Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another

Variation Among STRs Choosing which STRs: Significant statistical variation – but not too many. Freq. that are measured in pop. : Loc 1 -10%. Loc 2 – 10%; locus 1+2 -1/100. Random match with 13 primers (used now): 1/1013. (There are 6 billion people, ~ 6 x 109 people. ) Watch out for different racial types!

Multiplex PCR • Over 10 Markers Can Be Copied at Once • Sensitivities to levels less than 1 ng of DNA • Ability to Handle Mixtures and Degraded Samples • Different Fluorescent Dyes Used to Distinguish STR Alleles with Overlapping Size Ranges Most rxns: require 2 PCR (tubes) 7 or 8 primer pairs in one tube– need total of about 2 tubes for 13 different STRs. $20 -$25 per rxn in lab. $150 incl labor. Cost forensic up to $1000.

13 CODIS Core STR Loci with Chromosomal Positions TPOX D 3 S 1358 D 8 S 1179 D 5 S 818 FGA CSF 1 PO TH 01 VWA D 7 S 820 AMEL D 13 S 317 D 16 S 539 D 18 S 51 D 21 S 11 AMEL

D tells chromosome 21— Can tell Down’s syndrome. (No Down’s here. ) Human Identity Testing with Multiplex STRs Amelogenin protein is involved in tooth enamel and happens to be on sex chromosome – top: 2 peaks: x (106 bp) and y (112 bp); Bottom only 1 peak cause they have two X chromosomes. ® Two different individuals Amp. Fl. STR SGM Plus™ kit amelogenin D 19 106 bp 112 bp D 3 DNA Size (base pairs) D 8 TH 01 VWA D 21 D 16 D 18 D 2 FGA probability of a random match: ~1 in 3 trillion amelogenin D 3 D 19 D 8 VWA TH 01 Results obtained in less than 5 hours with a spot of blood the size of a pinhead D 16 D 21 FGA D 18 Simultaneous Analysis of 10 STRs and Gender ID D 2

FBI’s CODIS DNA Database Combined DNA Index System --all 50 states can upload their convicted felony and seq. of unsolved cases…. In Florida to convicted felon. • Used for linking serial crimes and unsolved cases with repeat offenders • Launched October 1998 • Links all 50 states • Requires >4 RFLP markers and/or 13 core STR markers • Current backlog of >600, 000 samples Except for police errors, and sufficient racial typing, it’s a done deal

Class evaluation 1. What was the most interesting thing you learned in class today? 2. What are you confused about? 3. Related to today’s subject, what would you like to know more about? 4. Any helpful comments. Answer, and turn in at the end of class.